Determination of Bone Density by DEXA Method Based on Bone Age and its Comparison with Chronological Age in Chronic Patients

Background and Objective: Given the growing awareness about the important role of children’s age in building bone for a person’s life, physicians need to assess bone health in high-risk children for bone density disorders more than before to optimize their bones’ density and prevent osteoporosis in future. The aim of this study was to evaluate bone density based on chronological and bone age. Materials and Methods: In this cross-sectional study, 80 Patients who have been referred for bone density to the Osteoporosis Centre of the Children’s Medical Centre over a one-year period (spring 98 to spring 99) were studied. Bone density was performed for all patients by using DEXA method. Results: The z-score mean chronological age for the lumbar spine was −0.8± 1.85 years and bone age was −0.58±1.64 years. The z-score mean chronological age for femoral bone was −1.6±1.02 years and bone age was −1.32± 1.4 years. Conclusion: Results showed that in all patients, the difference in the mean Z score of chronological age and bone age of the spine between patients was not significant but for femur was significant. Also, use of corticosteroids leads to significant difference between the two age groups’ z-score in femur and spine.


INTRODUCTION
Given the growing awareness of the important role of infant age in formation of bone for life, physicians consider it more important than ever to assess bone health in children who are at risk of bone density disorders or even in healthy ones, in order to help their bones to be optimized and prevent osteoporosis by increasing density. 1 Awareness of the patient's growth potential is one of the important points to be successful in various treatments. So, some indicators such as age, sexual maturity, skeletal growth stages, dental development stages, height, and weight are used to evaluate the growth stages. One of the most important growth indicators of people is their bone density. 2 Although the diagnosis of osteoporosis is not based solely on measuring bone mineral density during growth, it can be helpful in following the patients with primary and secondary osteoporosis. 1 According to the World Health Organization (WHO), osteoporosis is defined as a decrease in bone density by 2.5 points of the standard deviation from the mean maximum bone density in young and normal individuals of the community (T-score <-2.5). The decreased bone density between -1 to -2.5 points, less than mean maximum bone density in young and normal individuals of the community is called osteopenia, and the higher points would be considered normal (T-score < -1). It is noteworthy that osteoporosis in men under 50 years of age and premenopausal women and children is diagnosed based on the presence of fracture fragility or low Z-Score along with other risk factors of fracture. In cases that the Z-Score is less than -2, the term "low bone density" is used, and if it is greater than -2, the term "within the expected range" is used. 3 Another indicator of growth is a person's age, which may be different ages in a person's body, such as chronological age, height age, bone age, and so on. There are significant differences in children' development with the same chronological age, which indicates the need to understand physiological or biological age. Physiological age is the rate of progress toward puberty that can be estimated by dental, skeletal, sexual, or physical maturity. 4 The classical method of bone age assessment is based on recognizing the apparent changes in radiography and maturity indicators in left hand radiography, which is measured in comparison with a reference atlas. 5 The bone age can be used as a diagnostic or following method in patients suffering from growth or puberty disorders, and in some cases as a prognosis predictor for a person's final height. A child's bone age indicates the stage of biological and structural maturity better than the chronological age. Hand and wrist radiography is the most common method for calculating bone age. Automated methods for evaluating hand and wrist radiography have also been developed that reduce the technician based effects (on the study) compared to the manual method. 6 Research has shown that there is a significant correlation among chronological age, puberty and skeletal age. 7 In some cases, specifically chronic diseases and diseases need for steroid therapies, such as cystic fibrosis, Juvenile rheumatoid arthritis, and thalassemia, the development of the growth and height might be affected by the disease and therapies. 8 International guidelines recommend assessing the bone density in children with particular chronic diseases. Given that most of these children suffering from growth disorders, short stature and disproportionated bone age to the chronological age, so it is difficult to evaluate the bone density status. The DEXA method is currently used to assess bone density. DEXA can be used to estimate the exact structure of the body with minimal patient cooperation. Accurate interpretation of the obtained data by DEXA in children requires attention to bone size, stage of maturity, skeletal maturity, race, and body composition. 9 Low bone density in children according to the Z-score criteria is defined as less than -2SD. In screening, bone mineral density (BMD) of the spinal cord is more valuable than hip and femur bones. According to the texts and guidelines, in children with developmental disorders, this interpretation is more accurate based on skeletal growth with bone age assessment. 10 It would be confusing for physicians if any of these points not to be considered and might lead to incorrect interpretation, diagnosis, and treatment in these patients. At the moment, these points are not considered in bone densitometry centres. Other factors contributed to bone density disorders, including malnutrition, chronic infection, vitamin D deficiency, hypogonadism, delayed puberty, and decreased physical activity. 11 This study aimed to evaluate bone density based on chronological age and compare it to bone age in chronic patients referred to the bone densitometry centre of the Children's Medical Centre Hospital.

Study design and participants
This prospective cross-sectional study has been done on 80 patients who referred to the bone densitometry centre of Children' Medical Centre over a period of one year (July 2019 to July 2020). Along with DEXA method bone densitometry in patients, left wrist radiography was also performed for determination of bone age and to determine Z-score based on chronological and bone age.

Inclusion and exclusion criteria
Patients with a history of oral or long-time injectable corticosteroid use, referred rheumatologic patients, referred nephrotic syndrome patients, post-transplantation patients, patients after cancer recovery, CF patients, major thalassemia patients, patients with gastrointestinal and malabsorption problems and all chronic patients who had an indication to screening studies, were studied based on screening protocol. Patients under treatment with bisphosphonates, patients with disorders like skeletal dysplasia, imperfect osteogenesis, chronic renal failure, and congenital adrenal hyperplasia were excluded from the study.

Data collection and statistical analysis
A questionnaire containing demographic information, type of disease, duration of illness, drug history, history of fractures, bone pain, physical activity, and calcium and vitamin D intake, was completed for all patients. Also, serum level of calcium and vitamin D metabolites were checked for all patients if had not checked before. BMD of the lumbar spine (L2-4) using X-ray energy dose (DEXA) ( MA) was determined and expressed in grams per square centimetre. The low-density algorithm was used for values below 0.5 g / cm2. The coefficient variation for measuring repetition was 0.4% for 1 gr and 0.8% for 0.5 gr. The data obtained from this study were analysed with IBM SPSS version 22 software. Chi-Square tests were used to analyse the qualitative data between two groups and, if necessary, Fisher exact test was used. Paired-t test and, if necessary, Mann-Whitney U test were used to analyse quantitative data between two groups. Also, the statistical significance level was considered to be 0.05.

RESULTS
A total of 80 patients who referred to the bone densitometry centre of Children' Medical Centre from July 2019 to July 2020 were eligible for study. 42 of them (52.2%) were female and 38 patients (47.5%) were male. The mean weight of the patients was 36.12± 16.8 kg, the mean height was 137.94± 26.44 cm, the mean body mass index (BMI) was 18.52± 8.26 kg / m2, the mean age of them was 11.5±4.3 years (2.25-26.5) and their mean bone age was 10.27 ± 3.64 years. The mean Z-score of chronological age and bone age for spinal cord was -0.8±1.85 years and -0.58±1.64 years; respectively. The mean Z-score of chronological age and bone age for the femur was -1.6 ± 1.02 and -1.32 ± 1.4 years; respectively. The difference between the Z-score of chronological age and bone age was not significant for spinal cord, despite, it is statistically significant for the femur. In this study, 12 patients underwent bone marrow transplantation. Serum level of Alkaline phosphatase was significantly higher in these patients compared to the other ones. The mean Z-score of chronological age for spinal cord and Z-score of chronological age and bone age for the femur were significantly low compared to the other patients. In such cases taking corticosteroids, the difference of Z-score of the chronological age and bone age of spinal cord and femur was statistically significant. In patients who had a bone marrow transplantation, although the difference of the Z-score of chronological and bone age of the femur was significant but it was not significant for the spinal cord. In patients with cystic fibrosis (CF), the difference of the Z-score of bone age and chronologic age of spinal cord was significant, but it was not significant for the femur. In patients with rheumatologic disorders, the difference of Z-score of the chronological age and bone age of spinal cord and the femur was significant. In patients with Hematologic disorders, the difference of Z score of the chronologic age and bone age of the femur was significant, but this difference was not significant for the spinal cord. In these patients, the mean serum level of calcium was 10.12+10.43, the mean serum level of phosphorus was 3.56+ 0.976, the mean serum level of alkaline phosphatase was 608.4+ 262.97and the mean serum level of Vitamin D was 30.69+ 9.43. 58% of patients had Fanconi Anaemia and 4% of them had Scleroderma. Of all patients, 33 patients (41.3%) were in the first stage of maturity, 14 patients (17.5%) in the second stage of maturity, 14 patients (17.5%) in the third stage of maturity, and 14 patients (17.5%) in the fourth stage of maturity and 5 patients (6.3%) in the fifth stage of maturity. Two patients had limited daily movements and three of them had no movement, but the remaining (75 cases) had normal physical activity. Of all patients, 23 patients (28.8%) did not take calcium and 24 patients (30%) did not take vitamin D. Of all patients, 56 patients (70%) had a history of prednisolone intake, and 14 patients (17.5%) had a history of concomitant use of prednisolone and methotrexate. Bone pain was seen in 20 patients (25%) and 12 patients had a history of bone fractures (15%). The mean of laboratory, demographic, and bone density variables were not significantly different between the group taking prednisolone and the group that did not take prednisolone ( Table 1). The mean of laboratory and demographic variables showed a significant difference between the group that had physical activity and the group that did not. However, the mean Z-score of bone age and chronological age of the spinal cord and the Z-score of chronological age and bone age of the femur were significantly different between two groups ( Table 2). The mean of BMI, serum calcium, phosphorus, alkaline phosphatase, Z score of chronologic age of spina cord, Z score of chronologic age, and bone age of femur were not significantly different between the group with normal puberty and the group with puberty failure, but, the mean of height, mean Z score of bone age, weight, vitamin D, and Z Score of bone age of the spinal cord were significantly different between these two groups ( Table 3). Among children with different underlying diseases, the mean Z score of bone age and serum level of calcium were significantly different, but the difference between other variables in the healthy children group and the children with different underlying disease group was not significant. The mean of serum alkaline phosphatase, Z score of the chronologic age of spinal cord, Z score of chronologic age, and bone age of the femur were significantly different between the groups who underwent bone marrow transplantation and patients with other treatments, but the differences among the other variables were not significant ( Table 4). The difference of the mean of the demographic, laboratory variables, and bone density, between two groups of patients with bone pain and other patients was not significant ( Table 5).

DISCUSSION
The results of this study showed that the difference between Z-score of bone age and chronological age of femur in all chronic children was significant (mean Z-score of bone age and chronological age of femur and spine between patients with various underlying diseases (including cystic fibrosis, Rheumatologic, blood, immunological, neurological, endocrine, and nephritic syndrome disorders (no significant difference). There was also a significant difference between the Z age of bone age and the chronological age of the femur in patients with rheumatic diseases, blood disorders, patients with a history of corticosteroids and bone grafts. The difference between Z-score and bone age and chronological age of the spine was also statistically significant in patients with cystic fibrosis, patients with rheumatic disorder and patients with a history of corticosteroids.
Based on the results of this study, none of the variables were significantly different between the group with a history of prednisolone use and other patients. Also, children who did not have normal physical activity had significantly lower chronological and bone age Z-scores on both spine and femur than patients with normal physical activity. In patients who did not have normal puberty, vitamin D and Z-score levels had lower bone age in the spine than in other patients. Finally, patients who underwent bone marrow transplantation had higher alkaline phosphatase, lower chronological age of the spine, and lower chronological and skeletal age of the femur than other patients. Dr. Tabatabai et al. conducted a study to evaluate the rate of bone density determination by DXA method in the population of 10-20 years old in Tehran. In this study, bone density showed a positive correlation with age, height, weight, and puberty. They found that calcium intake was correlated with femoral bone density, while this correlation was not observed in spinal cord. 12 In 2005, a study by Pludowski et al. evaluated 151 healthy children aged 4-18 years and 61 children aged 5-20 years with bone disorders to compare the chronologic age and bone age obtained by DXA method and found a strong correlation between chronologic age and bone age in both genders. Finally, they concluded that the simultaneous use of chronological age and bone age provides valuable information about the status of bone maturity. 13 In 2019, a study by Zougbi et al. investigated the relationship between chronologic age and bone age calculated by DXA method and they found that, Z-score of bone age was less than chronologic age and finally concluded that bone age along with the chronologic age, helps to evaluate the status of patients with Duchenne muscular dystrophy who are under treatment by corticosteroids. 14 According to the findings of the present study, in general, the difference between chronological age and bone age was not significant among all patients for spinal cord, but it was significant for femur bone, which indicates that the Z score of the chronological age of femur can be used to assess the status of patients. The study also showed that in patients taking corticosteroids, the differences in chronological and bone age of the spinal cord and femur were statistically significant. Also, in patients who underwent bone marrow transplantation, the difference between the chronologic and bone age of the femur was significant, but this difference was not significant for spinal cord. In 2004, a study by Rita Ujhelyi et al. conducted to investigate the bone mineral density and bone homeostasis in cystic fibrosis patients and the changes over a two-year period, which showed that the bone age in adolescents was lower than the chronologic age. The BMD Z score of the lumbar and femoral neck area in each age group was less than normal. 15 In the present study, the mean TITLE Z-score of bone age of patients was 11 years and the mean Z score of bone age of spinal cord and femur was -1.534 and -1.7 years, respectively. In 2014, Janneke Anink et al. studied on bone age in patients with juvenile arthritis which showed that chronic inflammation along with glucocorticoid therapy and sedentary treatment exposed idiopathic juvenile arthritis (JIA) patients to an increased risk of growth retardation and decreased BMD.Use of Glucocorticoids associated with delay in bone age and female sex with low a score. 8 In 2019 a study by Marushko et al. evaluated the serum level of vitamin D and status of bone density in the adolescent with JIA and showed that serum level of vitamin D was low in 92% of patients and 60% of patients had a Z score below -2 in bone densitometry (16). In the present study, the mean bone age of the spinal cord and femur was 0.013 and -1.05, respectively and the mean level of vitamin D in these patients was 32.65 ng/mL. A study by Dr. Kosarian et al. conducted to investigate bone density and related factors in thalassemia major and intermedia patients. A cross-sectional study was performed on 125 patients with thalassemia major and intermedia who underwent blood transfusion in special centers in Sari-Iran and showed that according to the Z-score, 61% of the cases were osteopenic. The correlation of age with low BMD was significant. They concluded that bone density measurement should be routinely performed in these patients. 17 In 2003, a study by Dr. Shamshirsaz et al. aimed to evaluate BMD using the DEXA method in 212 thalassemia patients aged 10 to 20 years living in Tehran and determine the potential risk factors. There was not any significant difference in the severe decrease in bone density of spinal and femoral bones between boys (spinal 46.4%, femur 11.2%) and girls (spinal 54.7% and femur 10.4%). Reduced bone density found at 39.5% of the spinal bones and 37.5% of the femur bones. Patients with severely decreased bone density in the spinal bones were significantly older (P <0.001). The data obtained from this study confirmed a significant decrease in bone density in Iranian thalassemic patients. Although more research is needed, well-known prophylactic methods such as hormone therapy and rapid treatment of bone density disorders have been emphasized in thalassemic patients. 18 In this study, the mean bone age in patients with blood disorders (thalassemia major, Fanconi anaemic, acute lymphoblastic leukaemia [ALL] and acute myeloid leukaemia [AML]) was 10.88 years. The mean Z score of bone age of spinal cord and femur was -0.781 and -1.51; respectively. Serum levels of vitamin D and calcium were 8.9 and 28.6; respectively. In total, 12 patients underwent bone marrow transplantation in this study. Serum level of alkaline phosphatase was significantly higher in these patients than others, and the rate of bone age and the chronological age of the spinal cord and bone age of the femur were significantly lower than other patients. In 2007, Fatemeh Sayarifard et al. Studied on the bone density and diabetes in children and concluded that type 1 diabetes (DM) causes changes in BMD. The mean age was 12.5 years (4-14 years). Low BMD was detected in the lower normal range in 17 (15.2%) and 25 (22.3%) patients, respectively. There was a significant correlation between BMD and patient's age and age of diabetes diagnosis, IGF-1, HbA1c, and PTH, but only an increase in HbA1c level effectively predicted a decrease in BMD. 19 In 2011, J Feber et al. studied the effect of glucocorticoid in patients with nephrotic syndrome on bone age and bone density. They found that there was a reverse relationship between exposure to glucocorticoids and Z-score of spinal BMD and a small number of spinal lesions (8%). 20 In the present study, 8 patients had nephrotic syndrome. The mean bone age in these patients was 10.9 years. The mean of Z scores of chronological age and bone age According to some studies, there is no correlation between calcium intake in diet and bone mineral content in childhood. [21][22] However, in a prospective study on twins, the results showed that daily intake of 1000 mg of calcium significantly improved BMD. 23 Low bone density increases the risk of bone fractures and leads to increased mortality and morbidity in communities. 24 Low bone density in an adult can indicate a lack of maximum bone density during the early years of life. [25][26][27] Therefore, early diagnosis of at-risk individuals can reduce the risk of future bone fractures. During the years of growth, the amount of bone density is affected by the rate of skeletal system growing and increases with age. Bone minerals, which increase during the early years of life, decrease in the last years of life. 28 If bone density reaches to its desired level during childhood and adolescence, it would have a key role in preventing osteoporosis in the future. 29

CONCLUSION
The results of the present study showed that in all patients, the difference between Z-score of chronological age and bone age was not significant for spinal bone, but statistically significant for femur. The results also showed that corticosteroid intake could cause a difference between Z-score of chronological and bone age of the spinal cord and femur, and bone marrow transplantation could cause a difference between the Z-score of chronological age and bone age of the femur but have no effect on spinal cord. It is recommended that in infants with chronic diseases especially in infants were under corticosteroid therapy, in addition to chronological age we should use z-score of bone age in larger scale and with a larger number of samples over a longer period of time.